ALBERT

Beschreibung: Human cytomegalovirus (HCMV) infection can cause delayed leukocyte recovery after bone marrow transplantation and often associated with suppression of granulocyte/macrophage progenitor. Leukocyte lineage may be one of the major sites of HCMV infection. However, whether HCMV can interfere with CFU-GM formation and induce apoptosis in granulocyte/macrophage progenitor have not been well investigated. Human bone marrow mononuclear cells (Ficoll), promyelocyte cell line HL-60 and HCMV AD169 strain were co-cultured. Each bone marrow specimen was HCMV DNA and HCMV IgM negative by PCR and ELISA test. Our results showed that HCMV significantly inhibited the formation of CFU-GM as shown in two different concentrations of viral infection groups: 139.26 ± 5.42 (2×105pfu/ml), and 124.19 ± 8.82 (2×106pfu/ml) (colonies/2×105cells/ml, n=26). These were significant differences compared with the blank control group (P

Beschreibung: Abstract 2413 Poster Board II-390 Human cytomegalovirus (HCMV) can cause life-threatening infection in immunocomprimised individuals, such as patients undergoing intensive chemotherapy or bone marrow transplantation. Thrombocytopenia is one of the manifestations in active HCMV infection, which may be a consequence of viral suppression on megakaryopoiesis. The exact underline mechanisms remain uncertain. Our previous studies suggested that HCMV directly infects megakaryocytic progenitors and inhibits their proliferation. Colony-formation of HCMV-infected CFU-MK decreased in a dose-dependent manner (Blood, 2003, abstract). In present study, we explored the mechanisms further by using a phorbol 12-myristate 13-acetate (PMA) stimulating polyploidization to mimic the late stage of megakaryocytic differentiation and maturation in vitro. After co-culture of a megakaryocytic cell line CHRF-288-11 with HCMV AD169 experimental strain from day 0 to day 3 (multiple of infection, MOI=1), the polyploidization of megakaryocyte was determined by DNA content analysis using flow cytometry. Compared with negative control, the proportion of polyploidy (ploidy N ≥ 8) megakaryocytes decreased by 52%, 32% and 16% in HCMV-infected cell at day 3, day 6 and day 9 respectively. As a specific receptor for megakaryopoietic differentiation, the c-Mpl protein (TPO receptor) was also examined in CHRF-288-11 cell line. The proportion of c-Mpl positive cells showed a 23% decrease in HCMV-infected group in compared to the mock infection control (using ultralviolet treated HCMV) at day 5. In addition, apoptotic signals from megakaryocytic surface, cytoplasma and mitochondria were detected in HCMV-infected cells by flow cytometry with Annexin V, Caspase-3 and JC-1 assay. Compared to mock infection control at day 5, annexin-V positive cells population increased by 57%; active caspase-3 signal increased by 125% in viable cell population; and cell population with damaged mitochondial membrane showed a 5-times increase. In conclusion, our data demonstrated that: (1) HCMV inhibited megakaryocytic differentiation and maturation at late stage; (2) HCMV reduced c-Mpl positive cell population; (3) HCMV induced megakaryocytic apoptosis through intrinsic apoptotic pathway as shown by the functional alteration of mitochodial membrane, activation of caspase-3 and structural damage of outer cellular membrane. HCMV-induced thrombocytopenia is the consequence of multiple processes involving inhibition of megakaryocytic proliferation, differentiation, maturation and also increased megakaryocytic apoptosis. Disclosures: No relevant conflicts of interest to declare.